Journal: Advanced Science
Article Title: ASH1L in Hepatoma Cells and Hepatic Stellate Cells Promotes Fibrosis‐Associated Hepatocellular Carcinoma by Modulating Tumor‐Associated Macrophages
doi: 10.1002/advs.202404756
Figure Lengend Snippet: ASH1L‐mediated H3K4me3 modification increases CCL2 and CSF1 expression to recruit and M2‐polarize macrophages. A) Gene Ontology (GO) biological process enrichment analysis of the genes with down‐regulated H3K4me3 peaks at promoters in ASH1L knockdown SNU449 cells (cocultured with ASH1L knockdown LX2 cells). B) Venn plots (left) of genes with down‐regulated H3K4me3 peaks at promoters ChIP‐seq data and RNA‐seq data in SNU449‐sh1(LX2‐sh1) versus SNU449‐shCtrl(LX2‐shCtrl). Chord diagram (right) showing the core genes of signaling pathways. C) ChIP‐seq profile of differential H3K4me3 peaks in the CCL2 gene of SNU449 cells cocultured with LX2 cells as indicated. D) ChIP‐qPCR analysis of IgG, and anti‐H3K4me3 antibody at CCL2 and CSF1 promoters in SNU449 cells cocultured with LX2 cells as indicated. E) ChIP‐seq profile of differential H3K4me3 peaks in the CSF1 gene of LX2 cells cocultured with SNU449 cells as indicated. F) ChIP‐qPCR analysis of IgG, and anti‐H3K4me3 antibody at CSF1 and CCL2 promoters in LX2 cells cocultured with SNU449 cells as indicated. G) RT‐qPCR detected M2 markers expression in macrophages treated with the CM of cocultured primary hepatocytes and HSCs. The conditioned medium was supplemented with either PBS or 50 ng mL −1 of mouse recombinant proteins rCCL2 and rCSF1 (rCCL2 + rCSF1). H) Chemotactic migration assays of macrophages using the indicated CM of cocultured primary hepatocytes and HSCs ( n = 5). I) Representative images and quantification of tumor cell foci when cultured with the indicated CM from polarized macrophages induced by the cocultured SNU449 and LX2 cells ( n = 3). J) CFSE histograms detected the proliferation of CD8 + T cells cocultured with macrophages induced by the indicated CM of primary hepatocytes and HSCs ( n = 4). Data are presented as mean ± SD. P values were computed using the unpaired Student's t ‐test (D, F, G, H, I) and paired Student's t ‐test (J). ∗ P < 0.05, ∗ P < 0.01, ∗∗∗ P < 0.001. ns. not significant.
Article Snippet: Bulk liver RNA sequencing was performed by Novogene (Beijing, China).
Techniques: Modification, Expressing, Knockdown, ChIP-sequencing, RNA Sequencing, Protein-Protein interactions, ChIP-qPCR, Quantitative RT-PCR, Recombinant, Migration, Cell Culture
